Extracellular matrix cancer vaccine adjuvant

ABSTRACT

Compositions suitable for use as adjuvants in the preparation of vaccines, particularly those vaccines useful in the treatment of cancer, are provided. Methods for inhibiting tumor growth in an animal are also disclosed. Methods for immunizing an animal against cancer, such as prostate cancer, are also described. The adjuvants described are comprised of an extracellular matrix material, such as small intestinal submucosal (SIS) tissue. The preparations may take the form of sheets, gels, liquids (injectable), trocar, or other solid or semi-solid preparation. The invention provides for enhanced tumor inhibition of 2-fold or greater, compared to vaccine preparations without the extracellular matrix material, or from 4- to 5-fold, compared to preparations without the adjuvant promoting extracellular materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application makes reference to the following provisional U.S.Patent Application No. 60/730,379 entitled “Use of Extracellular MatrixMaterials as a Vaccine Carrier and Adjuvant”, filed Oct. 27, 2005, andto U.S. Ser. No. 11/209,766, entitled “Tissue Vaccines and UsesThereof”, filed Aug. 24, 2005. The entire disclosure and contents of theabove applications are hereby incorporated by reference herein.

STATEMENT OF JOINT RESEARCH AGREEMENT

In compliance with 37 C.F.R. §1.71(g) (1), disclosure is herein madethat the claimed invention was made pursuant to a Joint ResearchAgreement as defined in 35 U.S.C. 103 (c) (3), that was in effect on orbefore the date the claimed invention was made, and as a result ofactivities undertaken within the scope of the Joint Research Agreement,by or on the behalf of the University of Notre Dame and Cook Biotech,Inc. (West Lafayette, Ind.).

BACKGROUND

1. Field of the Invention

The present invention relates generally to cancer vaccines that includean adjuvant, and to cancer vaccine adjuvants alone. In particular, theinvention relates to cancer vaccine adjuvants derived or obtained atleast in part from biological tissues, particularly extracellular matrixmaterials, such as from the small intestinal mucosa. The invention alsorelates to the field of methods for immunizing an animal against cancerusing a cancer vaccine preparation that includes an extracellular matrixtissue-derived adjuvant. The invention also relates to the field ofmethods for preparing cancer vaccine adjuvants, as a method forpreparing a cancer vaccine adjuvant from extracellular matrix tissue forvaccines to immunize an animal against cancer, particularly prostatecancer, is provided.

2. Related Art

Vaccination for the treatment of cancer is receiving increasingattention. Vaccines for melanoma, prostate and breast cancers haveundergone development to include human clinical trials. Most of thesevaccines utilize specific proteins to directly immunize the patient orto pulse harvested dendritic cells prior to infusion into the patient.Some trials have also used inactivated allogenic cancer cells grown invitro.

In general, cancer vaccines have been administered without an adjuvantor with specific cytokines included as adjuvants. An adjuvant is definedas a compound which enhances the immune response to a vaccineimmunogen(s).

There have been some reports of the use of a mycobacterial adjuvant withnormal non-malignant cells. For example, use of human prostate cells inthe treatment of prostate cancer is described in U.S. Pat. No. 6,972,128(Dalgleish et al.). In particular, an allogeneic immunotherapeutic agentcontaining immortalized normal (non-malignant) human prostate cells(replication incompetent) is described. A mycobacterial adjuvant wasused with a non-malignant murine melanoma cell preparation in a vaccinesuitable for intra-dermal injection. These preparations were reported toprovide some protection against murine tumor cell growth.

A combination of aluminum hydroxide and aluminum phosphate (collectivelyreferred to as alum) is currently used in commercial vaccines asadjuvants for human and veterinary applications (11, 12). The efficacyof alum in increasing antibody responses to diphtheria and tetanustoxins is well established and HBsAg vaccine has been adjuvinated withalum. While the usefulness of alum is well established for someapplications, it has limitations. For example, alum is ineffective forinfluenza vaccination and inconsistently illlicits cell mediated immuneresponse. The antibodies elicited by alum-adjuvinated antigens aremainly of the IgG1 isotope in the mouse, which may be optimal forprotection by some vaccinal agents.

Bacterial vaccines have also been described that include an adjuvant,typically alum. Because alum is particularly efficient at stimulatingTh2 antibody responses to co-administered immunogens, and becauseeffective cancer immunity relies heavily on Th1 cell-mediated immunity,alum is not typically included in cancer vaccines. Clearly, cancervaccination would benefit from a method to provide general enhancementof the immune response to cancer immunogens.

Noscapine has been described as an adjuvant for vaccines, as well as foruse in the treatment of tumors and cancer, in U.S. Pat. No. 7,090,852.Noscapine is an alkaloid from opium, and is available as a commercialbyproduct in the commercial production of prescription opiates.

Recombinant, single immunogen cancer vaccines have also been described.One such product in Phase 3 clinical trials is the GVAX® vaccine (CellGenesys, Inc., South San Francisco, Calif.). This cancer vaccine is usedin patients with advanced-stage, hormone-refractory prostate cancer, andis comprised of two allogeneic prostate cancer cell lines that have beengenetically modified to secrete granulocyte-macrophage colonystimulating factor (GM-CSF). This hormone plays a role in stimulatingthe body's immune response to the cancer vaccine. The cells areirradiated for safety (3). Cancer vaccination with the GVAX product hasdemonstrated a median increases in survival in cancer patients receivingthe vaccine of approximately 7 months (4).

Though some studies have utilized specific cytokines as cancer vaccineadjuvants, such as GM-CSF in the GVAX vaccine (4), those cytokinestypically enhance only specific features of the immune response and maybe unstable outside of very controlled storage conditions (13, 14).

Pure soluble, recombinant and synthetic antigens, despite their bettertolerability, are unfortunately often much less immunogenic than live orkilled whole organism vaccines. Thus, the move towards the developmentof safer subunit vaccines has created a major need for more potentadjuvants. In particular, there is an urgent need for adjuvants capableof boosting cellular (Th1) immunity with a more acceptable toxicity.

Despite the description of over one hundred adjuvants in the scientificliterature, alum remains the only adjuvant approved for human use in theUSA (Petrovsky, 2006). Unfortunately, alum has no effect on cellularimmunity and is faced with increasing concerns regarding potential forcumulative aluminium toxicity. There is a major unmet need for a safeefficacious adjuvant capable of boosting cellular plus humoral immunity.

The prerequisites for an ideal cancer adjuvant differ from conventionaladjuvants for many reasons. First, the patients that will receive thevaccines are immuno-compromised because of, for example, impairedmechanisms of antigen presentation, non-responsiveness of activated Tcells and enhanced inhibition of self-reactivity by regulatory T cells.Second, the tumor antigens are usually self-derived and are, therefore,poorly immunogenic. Third, tumors develop escape mechanisms to avoid theimmune system, such as tumor immunoediting, low or non-expression of MHCclass I molecules or secretion of suppressive cytokines. Thus, adjuvantsfor cancer vaccines need to be more potent than for prophylacticvaccines, and consequently may be more toxic, and may even induceautoimmune reactions.

To heighten the immune response to cancer antigens, researchers oftenattach a decoy substance, or adjuvant, that the body will recognize asforeign. Such adjuvants are often proteins or bacteria which “trick” theimmune system into mounting an attack on both the decoy and the tumorcells. Other adjuvants act to stimulate specific effector cells withinthe immune system. Several adjuvants are described below:

Keyhole limpet hemocyanin (KLH) is a protein made by a shelled seacreature found along the coast of California and Mexico known as akeyhole limpet. KLH is a large protein that both causes an immuneresponse and acts as a carrier for cancer cell antigens (Bandandi, etal, 2006(52); Redfern et al, 2006(53)). Cancer antigens often arerelatively small proteins that may be invisible to the immune system.KLH provides additional recognition sites for immune cells known asT-helper-cells and may increase activation of other immune cells knownas cytotoxic T-lymphocytes (CTLs).

Bacillus Calmette Guerin (BCG) is an inactivated form of thetuberculosis bacterium. BCG is added to some cancer vaccines with thehope that it will boost the immune response to the vaccine antigen(Totterman, 2005(54); Mosolits, 2005(55)). It is not well understood whyBCG may be especially effective for eliciting immune response. However,BCG has been used for decades with other vaccines, including the vaccinefor tuberculosis.

Interleukin-2 (IL-2) is a protein made by the body's immune system thatmay boost the cancer-killing abilities of certain specialized immunesystem cells called natural killer cells. Although it can activate theimmune system, many researchers believe IL-2 alone will not be enough toprevent cancer relapse. Several cancer vaccines use IL-2 to boost immuneresponse to specific cancer antigens (Wei, 2006 (57); He, 2005 (56),Rousseau, 2006(58)).

Granulocyte Monocyte-Colony Stimulating Factor (GM-CSF) is a proteinthat stimulates the proliferation of antigen-presenting cells and hasbeen used as an adjuvant in a prostate cancer vaccine (Simons, 2006(59)).

SIS is a commercially available accellular extracellular matrix (ECM)preparation produced from porcine small intestinal submucosa. SIS is anaturally derived, extracellular matrix, that is not synthetic orcross-linked. A commercial form of this collagenous acellular materialis available from Cook Biotech, and is known by the trade name,“Oasis®”. In this product, SIS is taken from a biological source and isprocessed to remove all cells. This product is biocompatible and safefor human use.

SIS has found substantial utility as a tissue growth scaffold. Forexample, SIS has shown wide utility in urology (15-22), wound care andrepair (23-24), as an anal fistula plug (25), tendon repair, and bonehealing (26-27, 29, 31-33). Following implantation, SIS rapidly attractsmononuclear inflammatory cells followed by ingrowth of host tissue (FIG.1). In this way, SIS serves as a scaffold for tissue repair (26-28). TheSIS then becomes fully replaced by host tissue. Other extracellularmatrices, such as porcine renal capsule material, behave in a similarfashion to SIS (29-30).

Canine prostate cancer cells have been reported to maintain theirinvasive phenotype when grown on SIS in culture (44). Studies inLobund-Wistar rats have shown that SIS does not inherently promotegrowth of cancer in vivo (39). Despite these observations, SIS has notbeen proposed in any anti-cancer applications.

A need continues to exist in the medical arts for materials that may beused to enhance and/or improve existing clinical alternatives to thetreatment of cancer, particularly to improve existing forms of cancervaccines and cancer vaccine adjuvants with improved immunogenicity.

SUMMARY

The present invention was developed in part by the inventors'recognition of the robust inflammatory response invoked by a materialused in tissue repair known as SIS. From these observations, theinventors harnessed the inflammatory-provoking activity of SIS, andother materials prepared with SIS, in the design of a highlyimmunopotent cancer vaccine preparation and cancer adjuvant. Whilecompletely divorced from the field of tissue repair materials, thecrafting of cancer vaccine preparations using SIS and materials like itresulted in the design of the herein described cancer treatment andvaccination formulations prepared from extracellular matrix materials.

The present invention is unique in the respect that, among other things,it involves the modification and use of a three-dimensionalextracellular matrix material, SIS, and modified preparations thereof,to grow and expand tumor cells, and the use of these cultured tumorcells in an anti-cancer adjuvant.

Cancer Vaccine Adjuvant

In one aspect, the present invention provides an extracellular matrix(ECM) material, such as a modified preparation of SIS, FEM, RCM, orother appropriate extracellular matrix material of choice, as a cancervaccine adjuvant. In some embodiments, these preparations may bedescribed as essentially free of alum. In other embodiments, the ECMmaterials may be described as a modified preparation of SIS, FEM, RCM,or other extracellular matrix material of choice (diluted) about 2-foldto about 20-fold, or from 5-fold to about 10-fold. In some embodiments,a standard SIS material, such as that obtained from a commercial vendor,is diluted about 1-10 fold, and in this dilution, is particularly wellsuited for use as an injectable vaccine material. In particularembodiments, the extracellular material is diluted in a physiologicallyacceptable solution, such as saline.

Cancer Vaccine

In another aspect, the present invention provides a cancer vaccinecomprising a preparation of an extracellular matrix tissue together witha preparation of (replication incompetent) tumor cells. In someembodiments, the tumor cells are prostate cancer cells, breast cancercells, liver cancer cells, lung cancer cells, colon cancer cells, etc.In particular embodiments, the tumor cells are treated so as to renderthem replication incompetent by fixing the cells with glutaraldyhyde.This glutaraldyhyde preparation of tumor cells may then be mixed withthe extracellular matrix material, such as SIS.

In one aspect of the invention, there is provided a compositioncomprising an immuno-enhancing preparation of an extracellular matrixmaterial, particularly the extracellular matrix of the small intestinalsubmucosa (SIS) or tissue of the renal capsule. In some embodiments, theextracellular matrix comprises a menu of antigenic speciescharacteristic of porcine small intestinal submucosa. This preparationmay also be described as comprising a small intestinal submucosa tissuepreparation, or purified preparation thereof.

According to another aspect, there is provided a composition comprisingan adjuvant and a vaccine of interest. In some embodiments, the vaccineis a whole-cell vaccine. In some embodiments the vaccine may bedescribed as a cancer vaccine. In other embodiments, the vaccinecomprises an immunogenic amount of a tumor antigen preparation ofinterest; and a cancer adjuvant, wherein said cancer adjuvant comprisesa preparation characteristic of an extracellular matrix material, andwherein the immunogenic amount of the tumor antigen preparation ofinterest sufficient to stimulate a protective response in the presenceof the cancer adjuvant is less than the amount of the tumor antigenpreparation of interest sufficient to stimulate a protective response inthe absence of the cancer adjuvant.

Method of Preparing a Cancer Vaccine Adjuvant and a Cancer Vaccine

According to another broad aspect of the invention, there is provided amethod for preparing a cancer vaccine adjuvant. In some embodiments, themethod comprises obtaining an amount of small intestinal submucosa (SIS)or other extracellular matrix material of choice (FEM, RCM), andpreparing a processed preparation thereof suitable for use as a cancervaccine adjuvant in combination with an immunogenic amount of a wholecell antigen vaccine preparation, such as prostate cells.

In another aspect, the invention provides a method for preparing acancer vaccine. In some embodiments, the method comprises preparing acancer vaccine adjuvant as described, and combining the cancer vaccineadjuvant with an immunogenic amount of a cancer antigen of interest. Insome embodiments, the immunizing antigen of interest is a tumor cellpreparation, such as a prostate, lung, breast, colon, or other cancercell preparation. In some embodiments, the prostate cancer cellpreparation comprises prostate tumor cells harvested from an animal thathave been treated and/or processed with glutaradyhyde.

Methods of Treating/Inhibiting/Immunizing an Animal Against Cancer

According to yet another broad aspect of the invention, a method fortreating and/or immunizing an animal having cancer or at risk ofdeveloping cancer is provided. In some embodiments, the method comprisesimmunizing an animal against prostate, breast, colon, lung, or othercancer of interest, employing as antigen a tumor tissue comprising thespecific type of cancer cells of interest. In particular embodiments,the method provides for the treatment and/or immunization of a humanhaving or at risk of developing prostate cancer. The present inventionprovides for both a human vaccine and an animal vaccine.

In some embodiments, the method for treating prostate cancer employs acomposition comprising a vaccine, the vaccine comprising an adjuvantcomposed of an extracellular matrix (ECM) material together with atissue preparation, such as a glutaraldehyde-fixed xenogeneic tissuepreparation of prostate cancer cells. These preparations are found to bemore immunogenic than use of the glutaraldyhyde fixed xenogenic tissuepreparation without the extracellular matrix material adjuvant.

Method of Expanding a Tumor Cell Population

In yet another aspect, the invention provides a method for expanding apopulation of tumor and/or cancer cells in vitro. These cancer and/ortumor cells may then be used as an antigen of interest to be includedwith an extracellular matrix material adjuvant to provide a cancervaccine as described herein.

Clinical Cancer Treatment Preparations

In yet another aspect, the invention provides a variety of uniqueclinical cancer treatment preparations. In some embodiments, thesecancer treatment preparations may take the form of a gel, a sheet, or aninjectable preparation of an extracellular matrix material. Theinjectable preparations may be further described as suitable for i.v.administration.

The following abbreviations are used throughout the description of thepresent invention:

-   -   ECM—Extracellular Matrix;    -   FEM Fascia Extracellular Matrix Material;    -   GFT—Glutaraldehyde Fixed Tumor;    -   LW Rat—Lobund-Wistar rat;    -   MEM—Modified Eagle's Medium;    -   PAIII—Prostate Adenocarcinoma III Cell Line from LW rats;    -   RCM—Renal Capsule Material;    -   SIS—Small Intestinal Submucosa;

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in conjunction with the accompanyingdrawings, in which:

FIG. 1, according to one embodiment of the present invention, presents aremnant of SIS extracellular matrix material in a rat 28 days aftersurgical implantation. The remaining biomaterial is surrounded bymacrophages with occasional lymphocytes. Stained with H & E, 400×.

FIG. 2, according to one embodiment of the invention, presents a thinlayer of PAIII rat prostate adenocarcinoma cells along the edge of SISextracellular matrix material. PAIII cells had been co-cultured with SISfor three days. Stained with H & E, 400×.

FIG. 3, according to one embodiment of the invention, presents aphotomicrograph of SIS extracellular matrix material followingco-culture for three days with tumor cells obtained directly from asubcutaneous PAIII rat prostate adenocarcinoma tumor. The walls of theremnant blood vessel have been repopulated with cells and nuclei ofother cells can be seen within the substance of the SIS. Stained with H& E, 400×.

FIG. 4, according to one embodiment of the invention, presents aphotomicrograph of SIS extracellular matrix material followingincubation for three days in media but with no added cells. There are nonuclei present within the remnant vessel or the substance of the SIS.Stained with H & E, 400×.

FIG. 5, according to one embodiment of the invention, demonstrates theadjuvancy of GFT cell vaccine on SIS after three days of growth inculture. Cells harvested from PAIII rat tumors were grown on SIS forthree days. This cell population includes neoplastic epithelium,endothelial cells, fibroblasts and other connective tissue. SubcutaneousPAIII tumors were surgically resected and the GFT cell vaccine; GFT cellvaccine on SIS; or SIS without added cells placed onto the tumor bed.Rats were euthanized three weeks later and tumor weighed. Bars representmean group tumor weight (±standard deviation). A significant (P≦0.01)reduction in mean tumor weight was found in rats vaccinated with the GFTcell vaccine on SIS compared to all other groups.

FIG. 6, according to one embodiment of the invention, demonstrates theajuvancy of GFT cell vaccine on SIS after 28 days of growth in culture.Cells harvested from PAIII rat tumors were grown on SIS for 28 days.This cell population includes neoplastic epithelium, endothelial cells,fibroblasts and other connective tissue. Subcutaneous PAIII tumors weresurgically resected and the GFT cell vaccine; GFT cell vaccine on SIS;or SIS without added cells placed onto the tumor bed. Rats wereeuthanized three weeks later and tumor weighed. Bars represent meangroup tumor weight (±standard deviation). A nearly significant (P≦0.053)reduction in mean tumor weight was found in rats vaccinated with the GFTcell vaccine on SIS compared to rats vaccinate with the GFT cell vaccinealone; however the difference was significant (P≦0.01) compared togroups undergoing only resection or resection plus administration of SISwith no added cells.

FIG. 7, according to one embodiment of the invention, demonstrates theadjuvancy of SIS gel for the GFT cell vaccine in preventing tumorgrowth. Rats were vaccinated three times, seven days apart, with eitherSIS gel; SIS gel with GFT cells; GFT cells; or saline prior tosubcutaneous challenge with PAIII cells. Bars represent mean group tumorweights (±standard deviation). A significant (P≦0.01) reduction in meantumor weight was found in rats vaccinated with the GFT cell vaccine inSIS gel compared to all other treatment groups.

FIG. 8, according to one embodiment of the invention, demonstrates theadjuvancy of SIS gel and sheet SIS for the GFT cell vaccine in treatmentof PAIII prostate adenocarcinoma tumors following resection.Tumor-bearing rats were vaccinated three times, 7 days apart with eithersaline; SIS with no added cells; GFT cell vaccine; GFT cell vaccine inSIS gel; or GFT cell vaccine on SIS. Three days after the firstvaccination, tumors were surgically resected; 21 days after resection,animals were euthanized and tumors weighed. Bars represent mean grouptumor weights±standard deviation. Mean tumor weights for rats vaccinatedwith the GFT cell vaccine alone or in gel SIS were significantly(P≦0.05) less than rats vaccinated with saline or SIS with no addedcells. Mean tumor weight for rats vaccinated with the GFT cell vaccineon a sheet of SIS was significantly (P≦0.01) less than all othertreatment groups.

FIG. 9, according to one embodiment of the invention, demonstrates theeffect of SIS implantation on tumor recurrence. PAIII tumors recurred inall animals within 3 weeks of resection. Size of explanted tumors in thesham surgery group demonstrates a slower growth rate in tumors thatreach a critical size. *SIS overlay limited the size of the tumors thatrecurred (P=0.0009, versus tumor resection alone). Data are presented asmean±1 SD.

DETAILED DESCRIPTION

It is advantageous to define several terms before describing theinvention. It should be appreciated that the following definitions areused throughout this application.

DEFINITIONS

Where the definition of terms departs from the commonly used meaning ofthe term, applicant intends to utilize the definitions provided below,unless specifically indicated.

For the purposes of the present invention, the term “adjuvant” isdefined as a substance which enhances the immune response to animmunogen.

For purposes of the present invention, the term, “adjuvancy” is definedas the ability of an agent to enhance and/or promote the immune responseof animal to a particular antigen.

For the purposes of the present invention, the term “biosyntheticmaterial” is defined as a material that is in part or whole made up fromor derived from a biological tissue.

For purposes of the present invention, the term “biological tissue” isdefined as an animal tissue, including human, or plant tissue that is orthat once was (cadaver tissue, for example) part of a living tissue ororganism.

For the purposes of the present invention, the term “extracellularmatrix” is defined as a tissue derived or bio-synthetic material that iscapable of supporting the growth of a cell or culture of cells.

For the purposes of the present invention, the term “cancer vaccine” isdefined as any preparation capable of being used as an inoculationmaterial or as part of an inoculation material, that will provide atreatment for, inhibit and/or convey immunity to cancer and/or tumorgrowth.

For the purposes of the present invention, the term “immunize” isdefined as eliciting an immune response in an animal, both a humoralimmune response and a cellular immune response.

For the purposes of the present invention, the term “immune provokingamount” is defined as an amount of the antigen required to elicit animmune response in the animal.

DESCRIPTION

The description of the present invention is enhanced by the variousexamples that follow.

Example 1 Materials and Methods

The present example provides some examples of materials and methods thatmay be used in the practice of the present invention.

Small Intestinal Submucosa (SIS)

Small Intestinal Submucosa (SIS) was obtained from Cook Biotech, Inc.(West Lafayette, Ind.). The material was provided as a sterile,lyophilized sheet of extracellular matrix. Experimental grade materialwas provided for use in the present studies of an SIS preparation thatwas described as having been prepared by harvesting porcine jejunum andplacing 10- to 20-cm lengths into saline solution (31-33). Followingremoval of all mesenteric tissues, the jejunal segment was everted andthe tunica mucosa abraded using a longitudinal wiping motion with ascalpel handle and moistened gauze. The serosa and tunica musculariswere then gently removed using the same procedure. The remaining tissuewas disinfected with peracetic acid, rinsed extensively in high puritywater, and sterilized using ethylene oxide prior to implantation.

Renal Capsule Material (RCM)

RCM was obtained from Cook Biotech, Inc. (West Lafayette, Ind.).Briefly, renal capsule was dissected from mature pig kidneys immediatelyfollowing slaughter. It was thoroughly rinsed under running tap waterand disinfected using a dilute solution of peracetic acid in ethanol toremove potential contaminating bacteria and viruses (34). Followingdisinfection, the RCM was rinsed in high purity water to remove theacid, lyophilized into a sheet form, and subsequently sterilized priorto implantation using ethylene oxide gas.

PAIII Cells—

The PAIII cell line was derived from an authochthonous prostate tumor ofan LW rat. PAIII cells have been transplanted into LW rats for manypassages with no change in pattern of growth or disease. When PAIIIcells are transplanted subcutaneously into the flank of LW rats, large,metastasizing adenocarcinomas develop within 40 days, though initialtumors are palpable within 10 days. From the primary tumor, the PAIIIcells metastasize spontaneously to the lungs. PAIII tumors arehormone-independent and refractory to most treatments (35).

GFT Cell Vaccine

GFT cell vaccine was a glutaraldehyde-fixed tumor (GFT) suspension ofcells harvested from tumors grown in animals. GET cell vaccine wasprepared from tumor tissue (36). Specifically, three grams of asubcutaneous tumor tissue was harvested from a Lobund-Wistar rat andused in the vaccine preparation. The subcutaneous tumor had beenproduced by administering prostate adenocarcinoma cells isolated from anautochthonous, metastatic prostate adenocarcinoma in a LW rat (37).

The tissue was finely minced, repeatedly aspirated with a 1 cc syringe,and an aliquot drawn with a 20-gauge needle to eliminate largeaggregates to create a cell suspension in modified Eagle's medium (MEM).The cell suspension was incubated in 2.5% glutaraldehyde (v/v) at 37° C.for 120 minutes and then washed thoroughly with media to produce the GFTcell preparation.

Animals

LW rats obtained from a breeding colony maintained at the University ofNotre Dame were used for all studies. In this model, large tumorsdevelop subcutaneously following subcutaneous administration of 1×10⁶PAIII cells in approximately 99% of rats.

Subcutaneous Tumor and Tumor Resection Model

In this model, male, 3-4 month old LW rats are administered 1×10⁶ PAIIIcells subcutaneously into the flank. After 14-21 days, a palpable tumoris present, and by 40 days metastatic foci are present in the lungs. Forstudies involving resection, the animal is prepared for aseptic surgery.The visible tumor is resected, though the resection is not radical andsufficient tumor bed presumably remains, as tumor re-growth occurs in100% of untreated individuals.

Growth of Cells on SIS and RCM

Sheets of single-layer SIS or RCM are cut into 2×2 cm sections andplaced into Modified Eagle's Medium (MEM). PAIII cells (1×10⁶) or cells(1×10⁶) harvested directly from a PAIII subcutaneous rat tumor arelayered on the SIS or RCM and incubated at 37° C. To create the GFT cellvaccine on SIS, the SIS with attached cells then undergoesglutaraldehyde fixation (GFT) and washing. Glutaraldehyde fixationinvolves incubating cells in 2.5% glutaraldehyde (v/v) for 60 min at 37°C., and then washing with media.

Alum was purchased as Alhydrogel™, an aluminum hydroxide gel adjuvant(Brenntak Biosector, Frederikssund, Denmark).

Statistical Analysis—Results of survival versus non-survival followingchallenge with tetanus toxin were compared between groups using theChi-square test with two degrees of freedom. Differences were consideredsignificant when p≦0.05. Results for mean tumor weight were comparedbetween groups with the Wilcoxon rank sum test with significance reachedwhen p≦0.05.

Example 2 In Vivo Activity of Tumor Cell Vaccine and Cancer Adjuvant

The present example demonstrates the utility of the present invention asan effective cancer vaccine adjuvant in vivo.

Tumor cells were cultured on SIS. Following three days of growth, theSIS with attached cells were fixed with glutaraldehyde. Subcutaneoustumors grown in the flank of Lobund-Wistar rats which had beenadministered PAIII prostate cancer cells 10 days earlier were surgicallyresected.

Groups of 5 rats then underwent either no further treatment; treatmentwith glutaraldehyde-fixed tumor (GFT) cells applied directly on thetumor bed; treatment with glutaraldehyde-fixed (GF) SIS (without cells)applied on the tumor bed; or treatment with glutaraldehyde-fixed SIS(with cells) applied on the tumor bed. Three weeks later, after tumorshad re-grown in most rats, tumors were weighed with the followingresults:

-   -   No treatment=mean tumor weight of 11.64 grams    -   GFT cells=mean tumor weight of 10.54 grams    -   GF SIS=mean tumor weight of 12.31 grams    -   GF SIS+GFT cells=mean tumor weight of 4.77 grams

The addition of SIS to the OFT cell vaccine resulted in a greater than50% reduction in mean tumor weight and establishes that SIS is aneffective adjuvant for cancer (anti-tumor) vaccination.

Example 3 ECM Supports Cancer Cell Expansion

The present example demonstrates the utility of the invention forproviding a method for expanding a cancer cell population on anextracellular matrix material. The present example also demonstrates theutility of the invention for preparing a highly immunogenic populationof cells useful in a cancer vaccine preparation. In the case of cancer,it is likely that many key antigens are expressed by connective tissuematrix and involve interactions of neoplastic cells with theextracellular matrix. Cancer cell vaccines grown on an extracellularmatrix thus may be prepared according to the present example and used asimproved vaccine antigen compositions for vaccination.

1. Fascia Extracellular Matrix Material (FEM)

The present example demonstrates another example of the type ofextracellular matrix material that may be used in the practice of thepresent invention. The present example employs porcine fasciaextracellular matrix material (FEM).

Studies were conducted as described herein to examine the ability oftumor cells to grow on FEM. In these studies, it was demonstrated thattumor cells did grow robustly on the FEM material, comparable with thatgrowth supported on the SIS and RCM.

2. Expansion of Prostate Cancer Cells on SIS and RCM in Culture

Previous investigators have demonstrated the ability of pure cell linesto grow on SIS in vitro. For example, Badylak et al (38) showed SIS iscapable of supporting cultures of NIH Swiss mouse 3T3 fibroblasts,primary human fibroblasts, keratinocytes, endothelial cells, and anestablished rat osteosarcoma cell line. The present example demonstratesthat an extracellular matrix material preparation as describe hereinfrom SIS supports cancer cell growth. In particular, growth of aprostate cancer cell line and a mixed cell population harvested directlyfrom a subcutaneous tumor (the tumor having been produced by inoculationof rat PAIII cells into a Lobund-Wistar (LW) rat), are shown to grow onthe ECM materials under the conditions described here.

Sheets of single-layer SIS and RCM were cut into 2×2 cm sections andplaced into Modified Eagle's Medium (MEM). PAIII cells (1×10⁶), or cellsharvested directly from a PAIII subcutaneous rat tumor (1×10⁶), werelayered on the SIS and incubated at 37° C. for 72 hours, then fixed in10% neutral buffered formalin for 24 h, washed in 70% ethanol, placed inparaffin and sectioned at 4-5 μM. Sections were then stained withhematoxylin and eosin stain and examined for cell growth.

Samples which were incubated with pure PAIII cells demonstrated amonolayer of cell growth along the edges of SIS and RCM (FIG. 2). Incontrast, culture of cells harvested directly from tumors showed growthof cells along the edges of SIS and RCM. In addition, in themidsubstance; vascular structures were re-populated with cells (FIG. 3)compared to control SIS which had undergone incubation in media but withno cells added (FIG. 4). of Badylak et al (38) showed that ratosteosarcoma cells and endothelial cells grew only on the edge of theECM, while fibroblasts populated the ECM midsubstance. When co-cultured,keratinocytes and fibroblasts resulted in a distinct spatial orientationof the two cell types and early epidermal structures were formed.

This study demonstrated that prostate cancer cells and mixed cellpopulations harvested directly from tumors can be grown in culture onthree types (FEM, SIS and RCM) of ECM.

Example 4 SIS as a Vaccine Adjuvant to Prevent Regrowth of TumorsFollowing Surgical Resection

In earlier work, the present inventors described the ability ofglutaraldehyde-fixed tumor (GFT) cells harvested directly from a PAIIIrat tumor to prevent prostate cancer (36). Based upon this, the presentexample demonstrates that vaccination will inhibit the regrowth oftumors following surgical resection.

The present example demonstrates that an SIS/whole cell vaccineeffectively inhibits tumor regrowth following surgical resection anddebulking. Studies utilized the Lobund-Wistar (LW) rat prostate cancermodel which can be used to induce de novo prostate tumors by chemicalinduction, or it can be used to grow subcutaneous tumors followingimplantation of a prostate cancer cell line (PAIII cells).

Using the latter system, PAIII cells were administered subcutaneously togroups of LW rats. Fourteen days after administration of PAIII cells,tumors were surgically debulked and vaccines applied as follows:

Adjuvancy after Growth of Cells on SIS for 3 Days

Vaccine was prepared by allowing tumor cells harvested from asubcutaneous tumor to grow upon SIS in culture for 3 days, after whichthe material underwent glutaraldehyde fixation (GFT) and washing (GFTvaccine on SIS). Glutaraldehyde fixation involves incubating cells in2.5% glutaraldehyde (v/v) for 60 min at 37° C., and then washing withmedia. One group of 5 rats underwent only resection; one group had GFTcell vaccine applied to the tumor bed; one group had SIS applied to thetumor bed; and one group had GFT cell vaccine on SIS applied to thetumor bed. The results in terms of mean tumor re-growth (tumor weight ingrams±standard deviation) after 3 weeks are shown in FIG. 5 and were asfollows:

-   -   Resection only: 11.64±2.14 gm, 4/5 with lung metastases    -   SIS alone: 13.61±1.4 6 gm, 4/5 with lung metastases    -   GFT cell vaccine: 9.50±1.27 gm, 3/5 with lung metastases    -   GFT cell vaccine on SIS: 3.98±0.1.37 gm, 2/5 with lung        metastases

The tumors in rats vaccinated with the GFT cell vaccine on SIS weresignificantly smaller (P≦0.01) than those from rats vaccinated with theGFT cell vaccine alone and the control groups.

Adjuvancy after Growth of Cells on SIS for 28 Days

In a second study, cells were cultured on SIS for 28 days beforeimplantation. The results from this study are shown in FIG. 6 and are asfollows:

-   -   Resection only: 14.9 gm±2.12, 6/6 with lung metastases    -   SIS only: 15.6 gm, ±1.82 5/5 with lung metastases    -   GFT cell vaccine: 11.8 gm±1.46, 4/5 with metastases    -   GFT vaccine on SIS: 6.01 gm±1.17, 2/5 with lung metastases

Thus, the result is repeatable and demonstrates that the GFT vaccine onSIS also inhibited metastasis from the primary tumor to the lungs. Thesedata were not quite significant (probability of 0.053) due to the smallgroup size.

These data support the idea that efficacy of cancer vaccines is improvedby growth of vaccine cells on, or incorporation into, extracellularmatrices such as SIS.

Example 5 SIS Gel Acts as an Adjuvant for a Vaccine to Prevent Cancer

Because implantation of vaccines incorporated onto solid SIS matrixwould require incision of tissue, it may not be practical for allapplications. Thus, the present example demonstrates the utility of theinvention to provide a vaccine against cancer in a gel form using anextracellular matrix material, such as SIS, and the use of same as avaccine adjuvant.

SIS gel is supplied by Cook Biotech, Inc. (West Lafayette, Ind.) and isproduced from SIS material via an acid digestion and purificationprocess.

SIS gel was diluted 1:10 with sterile saline. Harvested,glutaraldehyde-fixed cells from PATH tumors were mixed into the SIS geldilution such that each 0.25 ml dose of SIS gel contained 5×10⁶ GFTcells.

Groups of ten (10) male LW rats were administered subcutaneously thefollowing:

-   -   0.25 ml of SIS gel;    -   0.25 ml of SIS gel+GFT cells;    -   0.25 ml of sterile saline containing 5×10⁶ GFT cells; or    -   0.25 ml saline.

Rats were vaccinated 3 times, 7 days apart. Seven days after the lastvaccination, all rats were challenged subcutaneously with 1×10⁶ PAIIIcells.

Three weeks after challenge with PAIII cells, rats were euthanized andtumors weighed. The results are shown as mean tumor weights (±standarddeviation) in FIG. 7 and are as follows:

-   -   Saline=1.02 g (±0.37), 5/6 rats with metastases to the lungs    -   GFT cell vaccine=0.86 g (±0.11), 6/10 rats with metastases to        the lungs    -   GFT cell vaccine in SIS gel=0.19 (±0.14), 1/10 rats with        metastases to the lungs

As can be seen in FIG. 7, treatment with the GFT cells alone resulted ina tumor size of approximately 0.86 g.+/−0.11 g., while treatment withGFT cells in the extracellular matrix material (SIS) in a gel formresulted in a tumor growth of approximately 0.19 g.+/−0.14 g., aboutone-fourth the size. Hence, the addition of the extracellular matrix gel(SIS) in a 1:10 dilution significantly adjuvinated the tumor growthinhibiting activity of the GFT cell preparation (fixed prostate cellvaccine antigen) about 4-fold to about 5-fold. Thus, it is demonstratedhere that the addition of an extracellular matrix material to acell-based cancer vaccine will significantly adjuvinate a tumor cellpreparation used as a vaccine, by 2-fold or greater.

Example 6 SIS Gel Acts as a Vaccine Adjuvant for the Treatment of Cancer

The present example demonstrates the utility of the present inventionfor providing an enhancement of immunity effective both as a preventivemeasure and as a therapeutic measure.

In the present example, groups of six rats were challengedsubcutaneously with 1×10⁶ PAIII cells to create tumors. Animals werevaccinated 3 times, 7 days apart; rats underwent surgical resection oftumors ten days after challenge, three days after the first vaccination.An additional group was included in which animals were vaccinated bysubcutaneous implantation of GFT cell vaccine on a sheet of SIS. Animalswere euthanized 21 days after tumor resection and tumors weighed.

The results from this study are shown in FIG. 8 and are summarized as:

Mean tumor weight (g) ± SD, Treatment Group Lung Metastases SalineControls 9.2 ± 2.2 g, 6/6 with metastases SIS alone 8.6 ± 1.8 g, 6/6with metastases GFT cell vaccine 5.8 ± 0.9 g, 4/6 with metastases GFTcell vaccine in SIS gel 5.0 ± 0.8 g, 3/6 with metastases GFT cellvaccine on SIS sheet 2.1 ± 1.1 g, 3/6 with metastases

These studies demonstrate that SIS gel has vaccine adjuvant activity andcan enhance protective immunity to cancer both before cancer cellchallenge and as an adjunct to surgical resection. This means that SISgel enhances immunity effective as a preventative measure (i.e., as avaccine), and as a therapeutic measure (FIG. 9).

Example 7 Safety of GFT Vaccine and ECM Adjuvants

The present example demonstrates the utility of the present invention asa clinically acceptable preparation for animal, including human,treatment. In particular, the present example demonstrates that thepreparations do not induce tissue damage, and does not result inautoimmune disease.

Both the GFT cell vaccine and SIS are safe to use in vivo. The presentexample demonstrates that repeated administration of the GFT cellvaccine failed to induce histopathologic or clinical disease in rats. Inaddition, the present example demonstrates that SIS did not promotetumor growth in vivo, and further demonstrated inherent inhibition oftumor growth in the LW rat tumor model. Furthermore, SIS is alreadyapproved by the U.S. Food and Drug Administration as a medical devicefor a variety of applications.

The present study demonstrates that repeated vaccination with thepresent preparations does not result in histological evidence ofautoimmune disease.

Groups of 10 three-month-old LW rats were each immunized and boostedmonthly for 12 months with either MEM or GFT cells. Freund's completeadjuvant was used for the initial vaccination, and Freund's incompleteadjuvant was used for booster vaccinations. Tissues were then harvestedat 15 months of age, fixed in 10% neutral buffered formalin, sectionedat 3-4 nm and stained with hematoxylin and eosin. All rats wereclinically normal for the duration of the study. Kidney, heart, brain,liver, testis, prostate/seminal vesicle, and spleen were examined andall found to be histologically normal.

These results demonstrate that repeated immunization with the GFT cellvaccines does not induce tissue damage suggestive of autoimmunity.

Example 8 SIS Does Not Promote Growth of Tumor Tissue When Placed InVivo

The present example demonstrates the utility of the extracellular matrixmaterial preparations as providing an anti-tumor activity with atumor/cancer cell preparation. The present example also demonstratesthat the present preparations do not themselves induce tumor and/orcancer growth.

Because cancer cells showed an ability to grow on SIS and RCM in vitro,it is important to determine if an ECM, such as SIS, would promote thegrowth of residual tumor cells if placed on the bed of a resected tumorin vivo.

To evaluate this, groups of 25 male LW rats, age 3 months, underwentinduction of subcutaneous PAIII tumors as described above. Animals werethen assigned to one of four different treatment groups:

-   -   sham surgery control;    -   physical encasement of the tumor with SIS (tumor was not        dissected from the underlying vascular bed);    -   complete tumor resection (all grossly visible tumor was        removed); or    -   complete tumor resection followed by overlying the resected        tumor bed with SIS (approximately 3×3 cm).

Three weeks later, rats were euthanized and the tumors weighed. Theresults (FIG. 9) show that SIS did not promote growth of PAIII tumorscompared to sham surgery or resection alone. Overlying of the resectedtumor bed with SIS led to a significant (P≦0.0009) decrease in tumorsize versus resection alone (39).

In culture, cancer cell lines and cancer tissue from harvested tumormaterial both grow rapidly on SIS and RCM. When inactivated byglutaraldehyde fixation, cancer cells and tissue grown on SIS preventregrowth of tumors following surgical resection. This effect is observedwhen cells are grown on SIS, and also when glutaraldehyde-fixed tumorcells are mixed into a gel form of SIS. Furthermore, SIS gel is shown toact as a vaccine adjuvant to prevent the development of cancer; that is,to stimulate protective immunity to challenge with live PAIII cells.

ECM materials, as demonstrated by SIS and RCM, both initiate a robustinflammatory response when implanted in vivo. While not intending to belimited to any particular theory or mechanism of action, it is believedthat any antigenic moieties carried along, whether adhered to the ECM orpresent in a gel or a particulate suspension, will be processed by theimmune system, thus possibly accounting for at least one theory by whichthe ECM may act as a vaccine adjuvant.

It is known that SIS contains a variety of bioactive species, includingTGF-β (41). While TGF-β can act as a tumor promoter in later stages oftumor progression, it functions as a tumor suppressor in earlytumorigenesis (42). Thus, administered at the proper time, such asfollowing resection, the utility discovered herein for SIS to inhibittumor growth may be utilized.

Example 9 Proposed Regimen for Clinical Application, Sheet SIS

The present example is provided to demonstrate the utility of thepresent vaccines in SIS for providing a treatment for cancer and/or toreduce/inhibit tumor growth by use of SIS in a sheet-like preparation.

Approach without Surgical Tumor Resection:

While vaccines based on an extracellular matrix have not been described,use of a prostate cancer vaccine comprised of inactivated allogeneicwhole prostate cancer cell lines has been described (Michael, etal)(2005)(47). In that study, monthly intradermal injections for 12months of 8×10⁶ inactivated whole cells were administered, the first twoin a standard adjuvant, alum, to patients with hormone-resistantprostate cancer. The adjuvant used in the first two doses administeredwas bacilli Calmette-Guerin. The first three doses were given at weeklyintervals, and once a month thereafter. This approach led tostatistically significant declines in PSA (prostate-specific antigen)velocity with no evidence of toxicity. Further, median time to a definedpoint of disease progression was increased to 58 weeks fromapproximately 28 weeks.

A whole cell prostate cancer vaccine together with a preparation of theextracellular matrix adjuvant (diluted 1×10 from a commercialpreparation, such as that commercially available from a vendor such asCook Biotech, Inc.) would be used according to the present inventionunder a clinical regimen wherein the vaccine would be administeredintradermally or subcutaneously on a monthly basis for approximately 12months.

Vaccine preparations which can be easily injected, such as thoseincluding SIS gel or a particulate form of SIS as adjuvant would beadministered by percutaneous injection.

A vaccine preparation which includes vaccine fixed on a sheet of SISwould be administered either percutaneously by trochar into thesubcutaneous space or, in other embodiments, by implantation via a smallincision made into the skin.

Approach with Surgical Resection:

Few studies have looked at the utility of vaccination in conjunctionwith surgical resection of a tumor. Pilla et at (2006)(49) administeredsubcutaneously tumor-derived heat shock protein gp96-peptide complexvaccine to advanced stage melanoma patients for up to four treatments,two weeks apart, following surgical resection. That approach resulted instabilization of disease in 11/18 patients post-surgically. Berd et al(1997)(50) administered an inactivated autologous whole cell vaccine ona weekly or monthly schedule to melanoma patients with clinicallyevident lymph node metastases; this approach resulted in survival ratessuperior to those resulting from surgery alone.

While no studies have looked at the utility of vaccination directly onthe tumor bed of a resected prostate tumor, nor the utility of a vaccineincorporated onto a solid-phase adjuvant such as an extracellularmatrix, the present examples demonstrate specific clinical useapplications of the vaccine. Some embodiments of the present inventionwill provide the vaccine incorporated onto a sheet of extracellularmatrix, and will be applied as a sheet directly onto the resected tumorbed at the time of surgery; or administered intradermally orsubcutaneously at a site beyond the tumor bed on a monthly basis. Asimilar approach used with a different vaccine is described by Berd etal (1997) (50) using a whole cell vaccine for the treatment of melanoma.

In other embodiments, a combination approach may be used in whichvaccination is made directly onto the tumor bed, and is applied at thetime of resection followed by booster vaccinations given intradermallyor subcutaneously.

The sheet vaccine would be administered percutaneously by trochar intothe subcutaneous space or, possibly, by implantation via a smallincision made into the skin. Vaccine preparations which can be easilyinjected, such as those including SIS gel or a particulate form of SISas adjuvant, would be administered by direct application of the materialonto the tumor bed and/or intramdermally or subcutaneously by injection.Bell et al. (2005) (65).

Example 9 Dermal Application of Vaccines

The present example demonstrates the utility of the invention forproviding a dermally-applicable formulation of the tissue based adjuvantcancer preparations.

While transdermal vaccination has been used for diseases associated withinfectious pathogens (Kenney, 2004(59); Skountzou, 2006 (60); Glenn,2006 (61), very few attempts have been made to apply this route ofadministration to cancer vaccines. Transcutaneous immunization was usedin mice by administering imiquiod, a cytotoxic T lymphocyte (CTL)activator, in an ointment applied to shaved skin (Rechsteiner, 2005(62)); this approach stimulated CTL activity in general and not againstany specific cancer antigen. Other investigators described an anti-tumorvaccine by delivery to mice of human carcinoembryonic antigen gene in anadenovirus vector via a thin film of vector placed onto the shaved skinand beneath a patch (Huang, 2005 (63)). This approach resulted inimmunologic resistance to challenge with murine mammary adenocarcinomacells.

According to use in the present invention, the cancer antigen ofinterest, such as a glutaraldyhyde fixed preparation of prostate cells,may be prepared in a formulation together with a gel form of theextracellular matrix material, SIS. In this formulation, the preparationmay be applied to an area to provide the anti-tumor effect.

Further features of embodiments of the present invention may be found inU.S. Provisional Patent Application No. 60/730,379 entitled “Use ofExtracellular Matrix Materials as a Vaccine Carrier and Adjuvant”, filedOct. 27, 2005, and to U.S. Ser. No. 11/209,766, entitled “TissueVaccines and Uses Thereof”, filed Aug. 24, 2005. The entire disclosureand contents of the above applications are hereby incorporated byreference herein.

Although the present invention has been fully described in conjunctionwith several embodiments thereof with reference to the accompanyingdrawings, it is to be understood that various changes and modificationsmay be apparent to those skilled in the art. Such changes andmodifications are to be understood as included within the scope of thepresent invention as defined by the appended claims, unless they departtherefrom.

BIBLIOGRAPHY

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

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What is claimed is:
 1. A composition comprising a tumor tissue cellsuspension comprising replication incompetent tumor cells, and anadjuvant comprising extracellular matrix from a non-tumor source.
 2. Acomposition comprising a prostate tumor tissue cell suspensioncomprising replication incompetent human prostate tumor cells, and anadjuvant comprising extracellular matrix from a non-tumor source.
 3. Thecomposition of claim 1 prepared by a method comprising: obtaining anadjuvant comprising extracellular matrix from a non-tumor source, saidextracellular matrix being capable of enhancing the immunogenicity of atumor tissue cell suspension; and combining said adjuvant with a tumortissue cell suspension of replication incompetent tumor cells.
 4. Thecomposition of claim 1 or 2 further defined as a pharmaceuticalpreparation.
 5. The composition of claim 1 wherein the replicationincompetent tumor cells are replication incompetent human tumor cells.6. An implantable preparation comprising the composition of claim 1 or2.
 7. The implantable preparation of claim 6 wherein the extracellularmatrix is in the form of a sheet, gel or particle.
 8. The implantablepreparation of claim 6 wherein the extracellular matrix is a gel.
 9. Thecomposition of claim 2 wherein the composition is essentially free ofalum.
 10. The composition of claim 2 wherein the prostate tumor cellsare rendered replication incompetent by glutaraldehyde treatment. 11.The composition of claim 1 wherein the extracellular matrix is in asheet, particle or gel form.
 12. The composition of claim 1 wherein thesource of the extracellular matrix is small intestinal submucosa.
 13. Amethod for inhibiting growth of a tumor in an animal comprisingadministering to an animal having a tumor the composition of claim 1,wherein the tumor tissue cell suspension comprises tumor cells obtainedfrom the animal to be treated.
 14. A composition suitable for treatingcancer comprising a tumor tissue cell suspension comprising replicationincompetent tumor cells, and an adjuvant comprising extracellular matrixfrom a non-tumor source.
 15. A prostate cancer treatment compositioncomprising a human prostate tumor tissue cell suspension comprisingreplication incompetent prostate tumor cells, and an adjuvant comprisingextracellular matrix from a non-tumor source.
 16. A method forstimulating an immune response in an animal in need thereof against acancer tissue comprising: administering to said animal a compositioncomprising a tumor tissue cell suspension of replication incompetenttumor cells, and an adjuvant comprising extracellular matrix from anon-tumor source.
 17. The method of claim 16 wherein the tumor tissuecell suspension is a prostate tumor tissue cell suspension.
 18. Acomposition comprising a replication incompetent tumor tissue cellsuspension and extracellular matrix, wherein said extracellular matrixis from a non-tumor source and in the form of a gel.
 19. A method forinhibiting tumor growth in an animal in need thereof comprisingadministering the composition of claim 18.